The goal of this research proposal is to identify the genes required for tubule formation. To accomplish this, I will test the role of candidate genes and pathways that may mediate tubulogenesis by using RNA interference (RNAi) methods on cells grown under the conditions of a new in vitro culture system: 2.5 dimensional culture (2.5D) of Mardin-Darby canine kidney (MDCK) cells. In preliminary experiments, I developed a culture method, 2.5D, that allows efficient MDCK tubulogenesis. Compared to hepatocyte growth factor-induced three-dimensional tubulogenesis, a common model system for investigation of in vitro tubulogenesis, the 2.5D model system shows more synchronized developmental intermediates. Hence 2.5D cultures are better suited for transcriptional profiling analysis. cDNA microarrays were used to measure the temporal changes of mRNA levels during tubulogenesis. The data were clustered to reveal developmental intermediate-specific genes on the basis of expression pattern. Classified genes were further analyzed with Cytoscape, a protein network and visualization tool loaded with the human protein interaction map. Notably, one subset of the identified genes whose expression patterns are similar is surprisingly well connected, indicating the existence of a group of co-regulated genes with similar protein functions. Interestingly, there is another coherent functional network, which may represent a key signaling pathway critical to tubulogenesis. However, it is still unknown how these genes affect tubule formation. To investigate the roles of these screened genes in tubulogenesis, I propose to block the function of the genes by RNAi, and examine how the depletion affects 2.5D tubulogenesis. Using confocal immunofluorescence microscopy, morphological changes caused by the RNAi will be analyzed qualitatively and quantitatively. In addition, I will examine how the functional blockade affects tubulogenic signaling pathways including extracellular signal regulated kinase (ERK) and signal transducer and activator of transcription activation (STAT). The potential signaling pathways found in my preliminary data will also be tested. Analyzing the molecular basis for normal tubulogenesis will contribute to understanding pathological conditions, such as congenital anomalies of the kidney and urinary tract, and autosomal dominant polycystic kidney disease and related cystic diseases. In addition, the cellular processes in tubulogenesis are related to those in tumorigenesis. Both tubulogenesis and tumorigenesis require a developmental program of invasive epithelial growth. Studying the basic biology of tubulogenesis may lead to a deeper understanding of the cellular and molecular mechanisms of cancer biology, and in particular metastasis.